The objective of this proposed study is to characterize the proteins that participate in the events leading from the association of the B subunits of cholertoxin with GM1 to the activation of adenylate cyclase by the Al subunit of choleragen. Due to the minute quantities of proteins involved, it is not feasible to purify proteins to homogeneity for complete characterization. For this reason, we propose to use a variety of affinity labelling techniques to covalently couple highly radioactive compounds to these proteins to aid in their identification. The molecular properties of the radioactive complexes will be determined by sodium dodecyl sulfate polyacrylamide gel electroporesis (SDS-PAGE), isoelectric focusing (IEF) and two dimensional gel electrophoresis. The following approaches will be attempted in our study. (i) Rat brain plasma membrane or neuroblastoma cells will be used to establish optimal conditions for cross-linking experiments. Radioactive ganglioside derivatives will be inserted into membrane, and cross linking reagents of different lengths and of different chemical properties will be used to permanently attach the ganglioside derivatives to the nearest neighboring proteins. The analysis of this radioactive complex will allow us to obtain important information about the proteins that are adjacent to the exogenously-acquired ganglioside in the membrane. (ii) Optimal conditions for the GM1-dependent attachment of radioiodinated choleragen to the membrane proteins will be determined. The affinity indirect cross linking technique will be employed to link radioiodinated toxin to membrane proteins. The proteins identified will be compared with those previously determined. This comparison will enable us to determine the similarity of the protein environments of endogenous and exogenously-acquired gangliosides. (iii) After we gain valuable information about the static picture of ganglioside-mediated association of choleragen to the membrane proteins, attempts will be made to explore the subsequent dynamic steps in the internalization of choleragen, which culminate in the activation of adenylate cyclase by the A1 subunit of choleragen. We would like to use either chemical or physical methods to block the choleragen internalization at different stages. Proteins in the internalization pathway may, therefore, be identified in this synchronization experiment.